The homeodomain is the highly conserved DNA-binding domain of a class of proteins that function as transcriptional regulators, specifying positional and temporal information in the commitment of embryonic cells to specific developmental pathways. An early step in the cascade of events associated with development is the sequence-specific binding of the transcriptional regulator (the homeodomain-containing protein) to a specific sequence or a specific set of sequences of DNA.The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [(HD(wt); residues 1-80 that encompasses the 60 residue homeodomain)] and those harboring mutations in helix III of the DNA recognition site [(HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC), ellipticity changes at 222 nm, and Trp fluorescence. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl. A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 50-500 mM NaCl to 50 mM Hepes pH 7.4 buffer. The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present, with a substantial increase in the unfolding entropy at Tm exhibited by HD(H52R) compared to that of HD(wt). Comparing HD(H52R/T56W) to HD(H52R), DSC results indicate that there is at most a small decrease in the entropy change at Tm. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model for HD(wt), HD(H52R), and HD(H52R/T56W). Also, the intrinsic tryptophanyl residue fluorescence of HD(wt) or HD(H52R) increases 1.6-fold during unfolding, which indicates that Trp48 is quenched by energy transfer in the folded form; phosphate binding produces an additional 50% quench of Trp48 which is relieved by DNA binding but not by thermal unfolding. Enthalpy values for the vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp duplex DNA have been determined by isothermal titration calorimetry (10-30 C) and found to be enthalpically controlled at 298 K. In contrast to the large negative heat capacity change observed for other specific protein-DNA interactions, the heat capacity change for the vnd/NK-2 homeodomain binding specific DNA is small and positive. For HD(wt), HD(H52R), and HD(H52R/T56W) binding sequence-specific duplex DNA, ordering of protein structure, solvent rearrangements, and possible DNA accommodations occur upon complex formation.